| Background Bisphenol A(BPA)is an environmental endocrine disrupt chemicals(EDCs),commonly used as plasticizers in the manufacture of various food packaging materials,causing widespread exposure in life,and has been shown to be significantly associated with the incidence of metabolic diseases such as obesity.Chronic low-grade inflammation is a hallmark of obesity,and NLRP3 inflammasomes are involved in the pathology of immune and inflammation-related diseases as a molecular platform that regulates pro-inflammatory cytokine secretion,and studies have shown that NLRP3 inflammasomes play a central role in obesity.Moreover,it has been found that long-term exposure of BPA under the induction of high-fat diet(HFD)can activate NLRP3 inflammasome in adipose tissue macrophages(ATMs),but the specific mechanism is unclear.NLRP3 inflammasome can be activated by a variety of pathogenic microorganisms and endogenous danger signals,such as ceramides produced by intracellular lipid metabolism(Ceramids,Cer).Metabolic disorders in the state of obesity can increase the level of Cer,and BPA can interfere with the process of lipid metabolism,thereby inducing lipid metabolism disorders and producing a"fattening effect".Studies have shown that BPA promotes de novo synthesis of Cer in adipose tissue.However,whether BPA in adipose tissue induces de novo synthesis of Cer to activate NLRP3 and the specific mechanism has not been elucidated.Objective In vitro experiments were used to elucidate the role of BPA in inducing de novo synthesis of Cer in adipose tissue to activate NLRP3 inflammasome in bone marrow macrophages(BMDMs),and the molecular mechanism of Cer activating NLRP3 inflammasome through the AMPK-ROS pathway was further explored by using ROS inhibitors and/or AMPKαl siRNA.Based on BPA’s ability to promote the occurrence and development of obesity,this study intends to provide a new idea for the prevention and treatment of metabolic syndrome associated with long-term exposure to BPA by targeting the immunometabolic pathway of Cer in tissues to activate NLRP3 inflammatories from de novo.Methods Mice were randomly divided into normal control diet(NCD)group,HFD group,HFD+1000nM BPA group,HFD+SPT inhibitor group,HFD+1000nM BPA+SPT inhibitor group,with 6 animals in each group.Mice are exposed to BPA by drinking water.Using SPT inhibitor group mice,epididymal adipose tissue was collected by intraperitoneal injection of SPT inhibitor polyconchicin 0.3 mg/kg every other day starting 13 weeks after BPA exposure,and cervical dislocation was sacrificed after 16 weeks.Each group took 100mg of adipose tissue and extracted lipids from adipose tissue with chloroform for the following experiments.Part Ⅰ:BPA induces activation of NLRP3 inflammasome in BMDMs by de novo-synthesized Cer in adipose tissueBMDMs in the femur and tibia of 6-week-old male C57BL/6J wild-type mice were isolated and cultured until the sixth day to identify purity by flow cytometry(FCM)for experiments.Under in vitro conditions,BMDM was first stimulated with 100 ng/ml LPS for 6h,and then cells were collected with lipids derived from the NCD group,HFD group,HFD+1000nM BPA group,HFD+SPT inhibitor group,HFD+1000nM BPA+SPT inhibitor group,and mouse adipose tissue from the HFD+1000nM BPA+SPT inhibitor group for 6 h.Western blot(WB)method detected the expression of NLRP3 activation related molecular proteins in cells.The expression level of mRNA of NLRP3 activation related molecules in cells was analyzed by real-time quantitative-polymerase chain reaction(qRT-PCR).Immunofluorescence(IF)detects NLRP3 protein expression in cells;Enzyme-linked immunosorbent assay(ELISA)detects IL-1β,IL-18 content in cell supernatant.BMDMs in the femur and tibia of 6-week-old male C57BL/6J wild-type mice were isolated and cultured until day 6 for experimentation.Under in vitro conditions,BMDMs were stimulated with 100 ng/ml LPS for 6 h before treatment with different concentrations(0,10,20,30 μmol/L)of exogenous C18 ceramide for 6 h to collect cells.qRT-PCR analyzed the expression level of mRNA in the molecule mRNA related to the activation of NLRP3 inflammasome in cells.IF detects NLRP3 protein expression in cells.Part Ⅱ:BPA-induced molecular mechanisms by which de novo synthesized Cer in adipose tissue activates NLRP3 inflammasome via the AMPK-ROS signaling pathwayBMDMs in the femur and tibia of 6-week-old male C57BL/6J wild-type mice were isolated and cultured until day 6 for experimentation.BMDMs were first stimulated with 100 ng/ml LPS for 6 h,and then lipids from mouse adipose tissue derived from NCD,HFD,HFD+1000 nM BPA,HFD+SPT inhibitor and HFD+1000 nM BPA+SPT inhibitor groups were treated with BMDMs for 6 h and cells were collected.WB detects protein expression of p-AMPKαl and AMPKαl in cells;FCM detects the production of ROS in cells.BMDMs in the femur and tibia of 6-week-old male C57BL/6J wild-type mice were cultured until day 6 for experimentation and divided into blank group and experimental group.The blank group did not do any treatment,and the experimental group was divided into HFD+1000nM BPA group,HFD+1000nM BPA+SPT inhibitor group,HFD+1000nM BPA+Si-N.C group,HFD+1000nM BPA+SPT inhibitor+Si-N.C group,HFD+1000nM BPA+AMPKα1 siRNA group and HFD+1000nM BPA+SPT inhibitor+AMPKα1 siRNA group.The Si-N.C and Si-AMPKα1 transfection groups were transfected for 24 h,then all experimental groups were stimulated with 100 ng/ml LPS for 6 h,and finally lipids derived from mouse adipose tissue in HFD+1000nM BPA group and HFD+1000nM BPA+SPT inhibitor group were treated for 6 h respectively to collect cells.IF detects ROS production in cells;WB detected the expression of NLRP3 inflammasome activation related molecular proteins in cells;qRT-PCR analyzed the expression levels of the molecule mRNA expression of NLRP3 inflammasome activation in cells.BMDMs in the femur and tibia of 6-week-old male C57BL/6J wild-type mice were cultured until day 6 for experimentation and divided into blank group and experimental group.The blank group did not do any treatment,and the treatment group was divided into HFD+1000nM BPA group,HFD+1000nM BPA+SPT inhibitor group,HFD+1000nM BPA+NAC group,and HFD+1000nM BPA+SPT inhibitor+NAC group.The NAC group using ROS inhibitors was first pretreated with 10 μg/ml NAC for 2 h,then all experimental groups were stimulated with 100 ng/ml LPS for 6 h,and finally lipids derived from mouse adipose tissue in the HFD+1000nM BPA group and HFD+1000nM BPA+SPT inhibitor groups were treated for 6 h respectively to collect cells.FCM and IF detect ROS production in cells;WB detected the expression of p-AMPKα,AMPKα and NLRP3 inflammatorysomes in cells.qRT-PCR analyzes the expression levels of NLRP3-activated molecules in cells.Results1.BPA induces de novo synthesized Cer in adipose tissue to activate NLRP3 inflammasome in BMDMsCompared with the AT-derived lipid-treated groups of mice in the NCD group and HFD group,the expression of NLRP3 in AT-derived lipid-treated BMDMs in the HFD+1000 nM BPA group was significantly increased,the expression of Caspase-1 and IL-1β proteins was significantly upregulated,the expression of NLRP3,ASC,Caspase-1 and IL-1β mRNA was significantly increased,and the contents of IL-1β and IL-18 in cell supernatant were significantly increased.The expression of the above indexes was significantly reduced in the BMDM treated with AT-derived lipids in the SPT inhibitor group.2.Exogenous C18 ceramide activates NLRP3 inflammasome in BMDMsCompared with the blank group,the expression of NLRP3 in exogenous C18 ceramide-treated BMDMs was significantly increased and increased dose-dependently.NLRP3,ASC,Caspase-1,and IL-1β mRNA expression were significantly increased.3.BPA induces de novo synthesis of Cer in adipose tissue to inhibit AMPKαphosphorylation and increase ROS productionCompared with the AT-derived lipid-treated groups of mice in the NCD group and HFD group,AMPKα phosphorylation and ROS production were significantly increased in AT-derived lipid-treated BMDM in the HFD+1000nM BPA group.AMPKα phosphorylation and ROS production were significantly reduced in AT-derived lipid-treated BMDMs in mice in the SPT inhibitor group.4.Reducing the expression of AMPKα protein,increasing BPA,inducing ROS production in BMDMs treated with de novo synthesis of Cer in adipose tissue,and activating NLRP3 inflammasome sAMPKα phosphorylation was significantly reduced in AT-derived lipid-treated BMDMs in the HFD+1000nM BPA group,ROS production was significantly increased,and NLRP3,ASC,Caspase-1 and IL-1β mRNA expression were significantly increased.Compared with AT-derived lipid-treated BMDMs in HFD+1000nM BPA group,after AMPKα1 siRNA transfection,the expression of AMPKα protein was significantly reduced or even almost not expressed,ROS production was significantly increased,and the expression of molecules related to NLRP3 inflammatory activation was significantly increased.5.Reducing ROS production inhibits BPA induction of de novo synthesis of Cer in adipose tissue to activate NLRP3 inflammasome in BMDMs,but does not affect the expression of AMPKα proteinCompared with the blank group,the phosphorylation expression of AMPKα in AT-derived lipid-treated BMDM in the HFD+1000nM BPA group was significantly reduced,the production of ROS was significantly increased,the expression of NLRP3,Caspase-1 and IL-1β proteins were significantly upregulated,and the expression of NLRP3,ASC,Caspase-1 and IL-1β mRNA was significantly increased.Compared with the AT-derived lipid-treated BMDM in the HFD+1000nM BPA group,ROS production and expression of NLRP3 activation were significantly reduced after NAC pretreatment,but the phosphorylation expression of AMPKα was not affected.Conclusions1.BPA induces de novo synthesis of Cer in adipose tissue to activate NLRP3 inflammasome in BMDMs.2.BPA induces de novo synthesis of Cer in adipose tissue,which inhibits AMPKαphosphorylation,causes increased ROS,and activates NLRP3 inflammasome in BMDMs. |
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